Lipid homeostasis is maintained through a complex network of hormonal, neuronal and environmental regulators. Inability to modulate lipid metabolism to maintain homeostasis is a hallmark of many disease states including metabolic syndrome, obesity, diabetes, cardiovascular disease and some cancers. Together, these diseases are the leading causes of morbidity and mortality in the United States and most other developed countries. Specific metabolic disturbances in lipid metabolism result in elevating the levels of circulating free fatty acids, which in turn lead to increased fatty acid internalization and ectopic accumulation of triglycerides. The correlation between chronically elevated plasma free fatty acids and triglycerides with the development of obesity, insulin resistance and cardiovascular disease has led to the hypothesis that decreases in pancreatic insulin production, cardiac failure, arrhythmias, and hypertrophy are due to aberrant accumulation of lipids in these tissues. The proposed work addresses how fatty acids traverse the plasma membrane and how they are trafficked into downstream metabolic pools. These are especially important questions as the underlying biochemical mechanisms, which govern the transport of fatty acids into the cell and trafficking into discrete metabolic compartments are poorly defined. An understanding of the mechanism(s) leading to cellular uptake of free fatty acids is essential to prevent and combat lipotoxicity leading to the pathologies listed above. Previous work has shown that one process driving fatty acid transport is vectorial acylation, where specific fatty acid transport (FATP) isoforms alone or in concert with specific long chain acyl CoA synthetase (Acsl) isoforms function in the concomitant transport and activation of fatty acids. It is hypothesized that specific isoforms of FATP and Acsl function in the vectorial acylation of different classes of fatty acids (saturated, monounsaturated, polyunsaturated and highly unsaturated) across the plasma membrane and direct their trafficking into discrete metabolic pools. This hypothesis will be tested by completing experiments detailed in the following Specific Aims: [1] Define the contribution of the different FATP and Acsl isoforms in the vectorial acylation of exogenous fatty acids in mammalian cells;[2] Establish whether the FATP and Acsl isoforms function in the selectivity and specificity of fatty acid trafficking;And [3] Distinguish and discriminate FATP-dependent fatty acid transport functions in studies employing compounds that inhibit transport identified in high throughput screens. These compounds are expected to provide mechanistic information useful to combat and prevent lipotoxicity. PUBLIC HEALTH RELEVANCE: Fatty acids are implicated in the development of obesity related illnesses including metabolic syndrome, type II diabetes and cardiovascular disease. The mechanisms causing the initiation and progression of these diseases are poorly understood. One method to prevent toxicity associated with fatty acid accumulation would be to inhibit fatty acid uptake and accumulation within cells. The planned studies will focus on the fatty acid transport proteins (FATP) and acyl-CoA synthetases (Acsl) to characterize their role in this process. This work will provide mechanistic details to understand how these proteins function in fatty acid uptake into a cell, will determine the specific roles of these proteins in the transport of specific classes of fatty acids, and will characterize small compound inhibitors that may be useful to develop drugs to combat fatty acid-related diseases.